臺灣博碩士論文加值系統

實驗背景
敘利亞先天性心肌病變倉鼠（Biobreeders strain Bio 14.6），
其肌質網功能有缺陷是經常被用來作為研究心臟衰竭的重要動物模
式。過去本實驗室的研究結果顯示，病變倉鼠 (32-52 週)的單一心
室肌細胞短暫鉀離子外流 (transient outward K+ current；Ito) 較小，
與動作電位期間較長的發現一致。但短暫內向離子流 (transient
inward current；Iti) 則較健康倉鼠小，與若干活體倉鼠實驗病變容易
引起心律不整的結果相反，有待進一步澄清。
實驗目的
1.探討健康及心肌病變倉鼠的左心室乳突肌和心室肌細胞,在灌流高
鈣低鉀溶液或連續電刺激下，何者易引起心律不整，以了解心肌病
變對激發性節律的影響。
2.若干基礎研究結果顯示，IGF-1 許多作用與胰島素 ( 0.1-10 µM) 類
似，可以改善擴張型心肌病變的結構及功能，但此作用是否與細
胞內鈣離子的調節有關，有待探討。本實驗研究胰島素 (1 µM)
對倉鼠心室肌組織及細胞離子流 (Iti、IK1)的影響，比較胰島素
作用下心肌病變倉鼠心肌的表現與健康心肌有何差異，以了解心
肌病變的細胞機轉。
實驗方法
一、離體心室組織實驗：
選用美國 Biobreeders 公司訂購的敘利亞雄性健康倉鼠10
隻 (F1B) 及心肌病變倉鼠20 隻 (BIO 14.6),和來自國家實驗動
物中心的健康倉鼠10 隻，飼養到週齡35 開始作實驗，以heparin
(5000 i.u./kg)由倉鼠頸部皮下注射，30 分鐘後再給予sodium
pentobarbital（50 mg/kg）進行腹腔注射，等動物完全麻醉後，沿
胸骨中線剪開迅速取出心臟置於經混合氣 (97% O2、3% CO2) 飽
和之 Normal Tyrode solution 中 (pH= 7.4)。之後，剪下左心室的
乳頭肌 (直徑1.5-2 mm)，應用傳統玻璃微電極 (內含3 M KCl)
連接於多頻道記錄器記錄左心室乳突肌 Normal Tyrode solution
及高鈣低鉀溶液中，每3-5 分鐘停止電刺激20 秒靜息後動作電
位及收縮力增強 (PRPC) 現象的變化，並觀察藥物 (如 insulin)
的作用。
二、單一心室肌細胞分離實驗：
取52-60 週心肌病變的倉鼠心臟，置於室溫下100 ﹪氧氣
飽和 HEPES-Tyrode solution 中。將膠管一端套入主動脈出口處，
並以細線固定，另一端連接 Langendorff 灌流柱上，進行冠狀動
脈灌流的細胞分離實驗。分離出心室肌細胞，儲存於室溫下
的高鉀溶液中，然後逐步將溶液換HEPES-Tyrode solution，
以恢復生理狀況下的鈣離子濃度。
三、單一心室肌細胞的電生理實驗：
在倒立顯微鏡下觀察所有的細胞，選取橫紋清楚且靜止無自
發性收縮之單一心室肌細胞，然後利用 whole-cell patch-clamp
的方法，觀察健康及心肌病變的敘利亞倉鼠，予 1 µM 胰島素之
前後動作電位與各離子電流變化。
實驗結果
1. 組織方面: 心肌病變倉鼠較健康倉鼠，在高鈣低鉀溶液中易引起
DAD 及靜息後易有激發性節律的產生。
2. 單一心室肌細胞方面: 電位固定於 -40 mV 去極化至+40 mV，時
間維持0.5 秒，然後再極化回 -40 mV，連續20 次的刺激，在52-60
週的心肌病變倉鼠 (n=6) 全部都有產生Iti，而健康倉鼠 (n=7) 只
有2 個有產生Iti。
3. 健康倉鼠心室肌細胞加入 1 µM 胰島素後， DAD 幅度及 Iti 明顯
增加,但對 IK1 無增加的效果 。在心肌病變倉鼠心室肌細胞加入 1
µM 胰島素後，DAD 幅度明顯增加，而對Iti 的影響有出現增加或
減少的情形， 可能是由於個體的變異性。
結論
1. 這個實驗應用靜息後激發性節律及連續電刺激的方式，證實心肌
病變
倉鼠較健康倉鼠易傾向於發生激發性心律不整。
2. 由於在離體組織及單一細胞實驗的數據顯示有許多差異，所以未
來仍
需進一步的實驗去釐清。

Introduction
The hereditary cardiomyopathic strain of the Syrain hamster (Bio
14.6) had been extensively used as an experimental model for the study
of cardiomyopathy and heart failure (HF). It was assumed that, because
of the inherited defects in the cytosolic calcium (Ca2+
i) reuptake process
in the sarcoplasmic reticulum (SR), the myopathic myocytes would prone
to develop Ca2+
i overload and the subsequent triggered arrhythmia.
However, paradoxically, part of our previous experiments revealed that
the myopathic hamsters had a lower incidence of reentrant atrial
tachyarrhythmias and a smaller transient inward current (Iti) after
prolonged depolarizing pulses (＞1000 ms) in concomitant with a longer
APD and a reduced transient outward k+ current (Ito) in ventricular
myocytes.
Aims
The aims of the present experiment were:
(1) to study the ventricular muscles and cardiomyocytes from healthy
and cardiomyopathic hamsters under conditions of intracellular
calcium overload (perfusion with high [Ca]o-low [K]o solution or
after repeated electrical pacing);
(2) to examine the genesis of ventricular arrhythmias in the
absence or presence of insulin, a polypeptide known to modulate
intracellular calcium ([Ca2+]i) and increase contractile function of
myopathic hamsters.
Material and Methods
1. Preparation of hamster ventricular tissues :
Animals (35-40 week-old) were anesthetized with an intreperitoneal
injection of sodium pentobarbital (50 mg/kg). Papillary muscle
obtained from left ventricle and perfused in vitro at 37℃. Steady-state
(2 Hz) and post-rest (20s) action potentials were recorded by
microelectrode techniques and twitch force by a transducer.
2. Isolation of ventricular myocytes:
Male myopathic Syrian hamsters (Bio 14.6, 52-60 week-old) and
healthy hamsters were anesthetized with pentobarbital and the heart
quickly removed and immersed in HEPES-Tyrode solution. The hearts
were perfused in a retrograde manner via polyethylene tube connected
through the aorta and left ventricule into the atrium. The free end of the
polyethylene tubing was connected to a Langendorff perfusion column
for perfusion with oxygenated HEPES-Tyrode solution at 37℃. The
perfusate was replaced with oxygenated Ca2+-free HEPES-Tyrode
solution, then was replaced with containing collagenase (1 mg/ml) and
protease (0.01 mg/ml) finally. The piece of tissue was cut into fine
pieces and gently shaken in 20 ml of high-K storage solution until
single cardiomyocytes were obtained.
3. Electrophysiology study:
The isolated cells were placed in a 1-ml chamber mounted on the stage
of
an inverted microscope and superfused (at 3 ml/min) with
extra-cellular solution appropriate to each patch-clamp experiment.
Results
1. Ventricular papillary muscle obtained from 35-40 week-old
myopathic hamsters (vs. those from healthy hamsters) are prone to
develop signs of calcium overload: delayed afterdepolarization (DAD)
and post-rest triggered arrhythmias in the presence of high-[Ca2+]o
l o w - [ K + ] o p e r f u s a t e .
2. In ventricular myocytes isolated from 52-60 week-old myopathic
hamsters, all preparations (n=6) generate transient inward currents (Iti)
after repeated (up to 20 times) depolarizing pulses (from -40 to +40
mV for 500 ms every sec). In healthy preparations, only 2 of 7
m y o c y t e s
develop Iti and the Iti are smaller in size.
3. Insulin (1 µM), a polypeptide known to enhance Ca2+ influx and induce
positive inotropic response in myocardial cells, exaggerates DAD and
Iti but does not increase inward rectifier K+ currents (IK1) in
preparations from healthy hamsters. In myopathic ventricular muscles,
insulin increases (to a lesser extent) the DAD but may increase or
decrease Iti in myopathic myocytes due to difference in the age of
animals and individual variation.
Conclusions
1. With the experimental protocols adopted in the present study (repeated
depolarizing pulses, post-rest potentiation of triggered actitivity, etc.),
it has been demonstrated that preparations from myopathic hamsters
are prone to develop triggered arrhythmias as compared to the healthy
control.
2. Further experiments are required to clarify the discrepancies in results
between ventricular tissues and cardiomyocytes.

目錄-------------------------------------------------------------------------- Ⅰ
圖目錄----------------------------------------------------------------------- Ⅳ
表目錄----------------------------------------------------------------------- Ⅴ
中文摘要-------------------------------------------------------------------- Ⅵ
英文摘要-------------------------------------------------------------------- Ⅹ
第一章 緒言---------------------------------------------------------------- 1
第一節 心臟衰竭的介紹------------------------------------------- 1
第二節 心律不整的機轉------------------------------------------- 2
第三節 敘利亞心肌病變倉鼠------------------------------------- 4
第四節 離子流的簡介---------------------------------------------- 8
第五節 胰島素的作用---------------------------------------------- 9
第二章 研究目的---------------------------------------------------------- 11
第三章 材料與方法------------------------------------------------------- 12
第一節 實驗動物---------------------------------------------------- 12
第二節 離體左心室組織實驗------------------------------------- 12
第三節 單一心室肌細胞實驗------------------------------------- 14
(一) 細胞分離------------------------------------------------- 14
(二) 實驗用玻璃微電極製備------------------------------- 16
(三) 全細胞膜膜電位箝定法------------------------------- 16
(四) 電生理研究方法---------------------------------------- 18
1. 單一心室肌細胞短暫內向電流的測量--------- 18
2. 單一心室肌細胞內向整流型鉀離子流的測量 18
第四節 實驗藥物及溶液配製------------------------------------- 19
第五節 實驗數據與統計------------------------------------------- 20
第四章 實驗結果--------------------------------------------------------- 21
第一節 離體倉鼠左心室乳突肌實驗--------------------------- 21
(一) 基本參數-------------------------------------------------- 21
(二) 健康與心肌病變倉鼠的電生理特性比較----------- 21
第二節 單一心室肌細胞實驗------------------------------------- 24
(一) 基本參數-------------------------------------------------- 24
(二) 健康及心肌病變倉鼠之心室肌細胞的離子流比較 25
第五章 討論---------------------------------------------------------------- 37
第一節 胰島素對健康倉鼠與病變倉鼠心室肌作用的異同 38
第二節 胰島素對激發性心律不整的影響---------------------- 39
第三節 胰島素對單一心室肌細胞上的電生理變化---------- 40
第四節 胰島素對收縮力的影響---------------------------------- 41
第六章 結論---------------------------------------------------------------- 42
第七章 參考文獻---------------------------------------------------------- 43


















圖目錄

圖一、健康倉鼠予灌流 Normal Tyrode solution 加1 µM 胰島素
後對DAD及心律不整的影響------------------------------------ 27
圖二、心肌病變予灌流 Normal Tyrode solution 加1 µM 胰島素
後對DAD及心律不整的影響------------------------------------ 28
圖三、健康倉鼠及心肌病變倉鼠的心室肌細胞予1µM 胰島素後
對短暫內向電流的影響-------------------------------------------- 29
圖四、健康倉鼠及心肌病變倉鼠的短暫內向電流比較-------------- 30
圖五、健康倉鼠的心室肌細胞予1µM 胰島素後對內向整流型鉀
離子流的影響-------------------------------------------------------- 31
圖六、心室肌細胞內向整流型鉀離子流給予胰島素前後隨時間的
衰退減現-------------------------------------------------------------- 32











表目錄

表一、健康倉鼠及心肌病變倉鼠的參數比較-------------------------- 33
表二、健康倉鼠及心肌病變倉鼠的乳突肌予胰島素於正常Tyrode
溶液下穩定期動作電位及收縮力的比-------------------------- 33
表三、健康倉鼠及心肌病變倉鼠的乳突肌予胰島素於高鈣低鉀溶
液下穩定期動作電位及收縮力的比較-------------------------- 34
表四、健康倉鼠及心肌病變倉鼠的乳突肌予胰島素於高鈣低鉀溶
液下靜息後激發性心律不整的比較----------------------------- 35
表五、健康倉鼠及心肌病變倉鼠的心室肌細胞電容比較----------- 35
表六、健康倉鼠及心肌病變倉鼠的心室肌細胞電生理特性比較-- 36
表七、健康倉鼠及心肌病變倉鼠的短暫內向電流比較-------------- 36

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